This invention relates generally to an apparatus for screening and the pharmacological profiling of compounds modulating a cellular physiological response. This invention also relates to devices for rapid assessment of the properties of compounds that modulate the activities of cell surface receptors and ion channels. More specifically, this invention relates to methods and apparatus for detecting, evaluating and characterizing the ability and potency of substances to act as agonists or antagonists against receptors and ion channels localized on a cell surface membrane.
Biological cells contain receptor molecules located on their external membrane. The function of these receptors is to xe2x80x9csensexe2x80x9d the cell environment and supply the cell with an input signal about any changes in the environment. In eukaryotic organisms such cell environment is comprised of the neighboring cells and the function of the receptor is to allow cells to communicate with each other directly (the paracrine regulatory system) or indirectly (the endocrine regulatory system) thus achieving harmonized response of a tissue, organ or a whole organism. In prokaryotic cells, the surface localized receptors provide a means for detecting extracellular environment.
Having received such a signal, neurotransmitters, hormones, chemoattractant or chemorepellant substances for example, the surface localized receptors transmit this information about extracellular environment into the cell through specific intracellular pathways in such a way that the cell responds in the specific fashion to accommodate these changes. When there is an altered supply of the external signal molecules or an altered activity of the cell surface molecules, the cell response would be abnormal causing malfunctioning of a tissue or an organ.
In eucariotic cells, receptor molecules determine the selective response of the cell. Each type of receptor can interact only with a specific set of ligand molecules. For example, adrenergic receptors interact with adrenaline and noradrenaline, cholinergic receptors interact with acetylcholine, serotoninergic receptors interact with 5-hydroxytriptamine, dopamineergic with DOPA and so on. The cells derived from the different tissues invariably express specific sets of tissue receptors. Different types of receptors are connected to different signal transduction pathways. For example, nicotinic cholinergic receptor, upon binding acetylcholine molecule, directly activates sodium channel (Claudio et al., 1987, is incorporated herein by reference). G-protein coupled receptors activate enzymes of second messenger pathways, for example, adenylate cyclase or phospholipase C with subsequent activation of cAMP or phosphoinositide cascades (Divecha and Itvine, 1995, is incorporated herein by reference). Receptor tyrosine kinases activate cascade of MEK/MAPK kinases leading to cell differentiation and proliferation (Marshall, 1995 and Herskowitz, 1995, are incorporated herein by reference)]. Cytokine receptors activate JAK/STAT cascade which in turn can regulate other pathways as well as activate gene transcription (Hill and Treisman, 1995, is incorporated herein by reference).
Together with the receptors, the cell surface membrane carries ion pumps, ion transporters and ion channels. These molecular assemblies work in concert to maintain intracellular ion homeostasis. Any changes in the activity of these systems would cause a shift in the intracellular concentrations of ions and consequently to the cell metabolic response.
Ion pumps act to maintain transmembrane ion gradients utilizing ATP as a source of energy. The examples of the ion pumps are: Na+/K+-ATPase maintaining transmembrane gradient of sodium and potassium ions, Ca2+-ATPase maintaining transmembrane gradient of calcium ions and H+-ATPase maintaining transmembrane gradient of protons.
Ion transporters use the electrochemical energy of transmembrane gradients of one ion species to maintain gradients of other ion counterpart. For example, the Na+/Ca2+-exchanger uses the chemical potential of the sodium gradient directed inward to pump out calcium ions against their chemical potential.
Ion channels, upon activation, allow for the ions to move across the cell membrane in accordance with their electrochemical potential. There are two main types of ion channels: voltage operated and ligand-gated. Voltage operated channels are activated to the open state upon changes in transmembrane electric potential. Sodium channels in the neuronal axon or L-type calcium channels in neuromuscular junctions exemplify this kind of channel. Ligand-gated channels are activated to the open state upon binding a certain ligand with the chemoreceptor part of their molecules. The classical example of ligand-gated channels is nicotinic cholinergic receptor which, at the same time, is the sodium channel.
There are numerous methods for detecting ligand/receptor interaction. The most conventional are methods where the affinity of a receptor to a substance of interest is measured in radioligand binding assays. In these assays, one measures specific binding of a reference radiolabeled ligand molecule in the presence and in the absence of different concentrations of the compound of interest. The characteristic inhibition parameter of the specific binding of the reference radiolabeled ligand with the compound of interest, IC50, is taken as a measure of the affinity of the receptor to this compound (Weiland and Molinoff, 1981 and Swillens et al., 1995, are incorporated herein by reference). Recent advances in microchip sensor technology made it possible to measure direct interactions of a receptor molecule with a compound of interest in real time. This method allows for determination of both association and dissociation rate constants with subsequent calculation of the affinity parameter (F_gerstam et al., 1992, is incorporated herein by reference). While being very precise and convenient, these methods do not allow to distinguish between agonist and antagonist activity of the compound.
The type of biological activity of the compounds, agonist or antagonist, may be determined in the cell based assays. In the methods described in Harpold and Brust, 1995, which is incorporated herein by reference, cells cotransfected with a receptor gene and reporter gene construct, are used to provide means for identification of agonist and antagonist potential pharmaceutical compounds. These methods are inconvenient because they require very laborious manipulations with gene transfection procedures, are highly time consuming and use artificially modified cells. Besides, to prove that the agonistic effect of a particular compound is connected to the stimulation of a transfected receptor, several control experiments with a positive and negative control cell lines should be performed as well.
Most closely related to the methods of this invention are the methods described in Parce et al., 1994, which is incorporated herein by reference. These prior art methods use natural cells and are based on registering the natural cell responses, such as the rate of metabolic acidification, to the biologically active compounds. The disadvantage of the prior art is low throughput speed, each measurement point taking about three minutes. Another disadvantage of the prior art is the use of cells immobilized on the internal surface of the measuring microflow chamber. This leads to the necessity of using separate silicon sensors, or cover slips, with the cells adherent to them for each concentration point of the agonist or antagonist, for the receptors that undergo desensitization upon binding to the agonist molecule. This results in high variability of the experimental results.
Ionized calcium, unlike other intracellular ion events, e.g. changes in the intracellular concentrations of protons, sodium, magnesium, or potassium, serves as the most common element in different signal transduction pathways of the cells ranging from bacteria to specialized neurons (Clapham, 1995, is incorporated herein by reference). There are two major pools which supply signal transduction pathways in the cell with the calcium ions, extracellular space and the endoplasmic reticulum. There are several mechanisms to introduce small bursts of calcium into cytosol for signal transduction.
Both excitable and nonexcitable cells have on their plasma membrane predominantly two receptor classes, G-protein coupled serpentine receptors (GPCSR) and the receptor tyrosine kinases (RTK), that control calcium entry into cell cytoplasm. Both GPCSR and RTK receptors activate phospholipase C to convert phosphatidylinositol into inositol(1,4,5)-trisphosphate (InsP3) and diacylglicerol. InsP3 acts as an intracellular second messenger and activates specialized receptor that spans the endoplasmic reticular membrane. The activation of this receptor triggers release of calcium ions from the endoplasmic reticulum (Berridge, 1993, is incorporated herein by reference). The calcium ions can also enter the cytoplasm of excitable and nonexcitable cell from extracellular environment through specialized voltage-independent Ca2+-selective channels triggered by specific ligands. In nonexcitable cells, hyperpolarization of the plasma cell membrane also enhances entry of calcium ions through passive transmembrane diffusion along the electric potential. For example, opening of potassium channels brings the membrane potential to more negative values inside the cell, thus facilitating Ca2+ entry across the plasma membrane. Excitable cells contain voltage-dependent Ca2+ channels on their plasma membrane, which, upon membrane depolarization, open for a short period of time and allow inflow of Ca2+ from external media into cytoplasm. The endoplasmic reticulum membrane as well as plasma membrane of the excitable cells contains InsP3 receptors and Ca2+-sensitive ryanodine receptors (RyR) releasing Ca2+ from intracellular stores upon membrane receptor triggered phospholipase C activation or depolarization-induced short burst of Ca2+ entry into cell cytoplasm from extracellular media respectively.
It is well established that G-protein coupled serpentine receptors initiate Ca2+ mobilization through the activation of phospholipase Cxcex2 (Sternweis and Smrcka, 1992, is incorporated herein by reference) whereas tyrosine kinase receptors activate phospholipase Cxcex3 with subsequent intracellular Ca2+ mobilization (Berridge and Irvine, 1989, is incorporated herein by reference).
There are many plasma membrane G-protein coupled serpentine receptors, tyrosine kinase growth factor receptors and voltage- and ligand-regulated channels known to initiate intracellular Ca2+ mobilization.
Ca2+ plays an essential role in many functional processes of a cell. For example, Ca2+ affects the cell cycle (Means, 1994, is incorporated herein by reference) and activates specific transcription factors (Sheng et al., 1991, is incorporated herein by reference). Scores of receptors and ion channels use the Ca2+ signal to initiate events as basic as cell motility, contraction, secretion, division etc.
Increases in cytosolic and, consequently, in nuclear concentration of the Ca2+ can also be a cell death promoting signal. For example, prolonged increase in free Ca2+ activates degradation processes in programmed cell death, apoptosis, activates nucleases that cleave DNA and degrade cell chromatin, promotes DNA digestion by direct stimulation of endonucleases, or indirectly by activation of Ca2+-dependent proteases, phosphatases and phospholipases, resulting in a loss of chromatin structural integrity (Nicotera et al., 1994, is incorporated herein by reference).
A development of intracellular fluorescent calcium indicators (Grynkiewicz et al., 1985, is incorporated herein by reference) made it possible for intracellular concentration of free calcium to be measured directly in the living cell. Thus the ability to register changes in intracellular calcium concentration provide the means for monitoring effects of different compounds useful in treating various diseases, whose action is thought to be a result of an interaction with membrane receptors and ion channels.
With the advent of combinatorial chemistry approaches to identify pharmacologically useful compounds, it is increasingly evident that there is a need for methods and apparatuses capable of performing automated characterization of pharmacological profiles and corresponding potencies of the compounds in synthesized combinatorial libraries. This would enable the rapid screening of a large number of compounds in the combinatorial library the identification of those compounds which have biological activity, and the characterization of those compounds in terms of potency, affinity and selectivity.
It is an object of this invention to provide methods for screening and the quantitative characterization of potentially pharmacologically effective compounds that specifically interact with and modulate the activity of cell membrane receptors, ion pumps and ion channels using living cells.
It is an additional object of this invention to provide methods capable of characterizing an affinity of the active compounds to the binding sites of the cell.
It is another additional object of this invention to provide methods to distinguish between agonistic and antagonistic activity of the compounds.
It is yet another additional object of this invention to provide methods to determine the nature of the receptor, ion channel or ion pump entity which is sensitive to the active compounds discovered during the screening process.
It is yet another additional object of this invention to provide methods to characterize cell receptor pattern for particular cell source tissue.
It is yet another additional object of this invention to perform each of the above methods on each member of a series of cell types.
It is yet another additional object of the invention to determine the pattern of cell surface receptors expressed in one or more cell types.
It is yet another additional object of the invention to confirm that a test compound influences the activity of a particular receptor.
It is yet an additional object of the invention to determine the activity of a given receptor in a variety of cell types in which it is expressed.
It is a specific object of this invention to provide an apparatus for fulfillment of the objectives above.
It is yet another additional object of this invention to provide an apparatus for fulfillment of each of the objectives above for each member of a series of cell types.
At least some of these and other objectives are addressed by the various embodiments of the invention disclosed herein.
The present invention addresses the above and other needs by providing a method and corresponding apparatuses which allows the automated characterization of pharmacological profiles and corresponding potencies of compounds in synthesized combinatorial libraries. This enables the rapid screening of a large number of compounds in the combinatorial library, the identification of those compounds which have biological activity, and the characterization of those compounds in terms of potency, affinity and selectivity.
A variety of effects caused by the compounds to be screened may be detected and quantitatively characterized according to the present invention. Preferably, these effects include but are not limited to changes in intracellular concentration of ionized calcium, cAMP or pH, transmembrane potential and other physiological and biochemical characteristics of living cell which can be measured by a variety of conventional means, for example using specific fluorescent, luminescent or color developing dyes.
The present invention also includes methods of screening for agonist or antagonist activity of drugs, methods of characterizing their potency profiles, methods of identifying the receptor expression pattern of cell membrane (xe2x80x9creceptor fingerprintingxe2x80x9d) and methods of determining toxicity profiles for the compounds. In these methods, a steady flow of cells is mixed with flows of the compound and a standard substance. The effects of the compound alone and in mixture with the standard substance are measured and provide the means for pharmacological profiling of the compounds, drug screening and cell receptor pattern characterizing.
In a preferred embodiment of the invention, the compounds to be screened and standard agonist and antagonist substances are organized in a 96-well plate format, or other regular two dimensional array, such as a 48-well and 24-well plate format or an array of test tubes. In another preferred embodiment of the invention, the non-adherent cells are grown in a suspension of freely flowing cells by growing them in an appropriate cell cultivating system.
In another preferred embodiment of the invention, the naturally adherent cells which need attachment to a surface for their growth, are grown in the appropriate cell cultivating system containing commercially available micro spherical beads to which the cells adhere during the growth.
In yet another preferred embodiment of the invention, the naturally adherent cells which need attachment to a surface for their growth, are grown in the cell culture flasks with a subsequent detachment of the cells from the flask bottom with an appropriate detaching reagent.
In accordance with the present invention, either eukariotic or prokariotic cells can be used. The cells can be transfected with a gene coding to express a receptor of interest, for example, an orphaned receptor. In addition, the variety of compounds having biologically relevant activity may be used including but not limited to neurotransmitters, hormones, toxins, receptor activators and inhibitors, ion channels and ion pump modulators, irritants and/or drugs.
The cells grown in accordance with the preferred embodiments described above, are mixed with an appropriate fluorescent dye, for example FURA-2AM for measurements of concentrations of intracellular calcium or BCECF-AM for measurements of intracellular pH, and are incubated in the appropriate conditions to allow the dye to penetrate into the cell. The cells loaded with a dye are supplied to the apparatus. In the apparatus, the cells are successively mixed with a solutions of the compounds to be tested.
One aspect of the present invention is a method for identifying compounds having biological activity, comprising the steps of: (a) combining a homogeneous suspension of living cells with a test compound having an unknown cellular effect to form a test mixture, (b) directing the test mixture through a detection zone; and (c) measuring a cellular response of the suspended cells to the test compound as the test mixture is flowing through the detection zone. The method will often include the additional steps of: (d) combining a homogeneous suspension of the cells with a standard compound having a known effect on the cellular response of the cells to form a standard mixture; (e) directing the standard mixture through the detection zone; and (f) measuring the cellular response of the cells to the standard compound. In one embodiment, the standard compound and the test compound are simultaneously mixed with the cells in the combining steps, and the measuring step detects the known effect or an alteration of the known effect. The standard compound can be an agonist or antagonist of the cellular response. In one mode of operation, steps (a) and (d) are performed simultaneously; steps (b) and (e) are performed simultaneously; and steps (c) and (f) are performed simultaneously using a single suspension of the cells. In another mode of operation, steps (a), (b), and (c) are performed first, and then steps (d), (e), and (f) are performed, wherein the test compound is added together with the standard compound in step (d). If the cellular response is detected in step (c) to indicate that the test compound is active to generate the response, and the standard compound is an antagonist, then a decrease in the cell response from step (c) to step (f) is indicative that the test compound is an agonist of the known effect. If the cellular response is not detected in step (c), indicating that the test compound is not active to generate the response, and the standard compound is an agonist, then an alteration of the known effect detected in step (f) is indicative that the test compound is an antagonist of the know effect. Preferably, the method is performed automatically under the direction of a programmable computer on a plurality of test compounds and a plurality of standard compounds, and a successive series of antagonists are automatically added as the standard compound in step (d) if the cellular response is detected in step (c) to indicate that the test compound is active to generate the cellular response, whereby a decrease in the cellular response detected in step (f) is indicative that the test compound is an agonist of the known effect; and a series of agonists are automatically added as the standard compound in step (d) when the cellular response is not detected in step (c), whereby an alteration of the known effect detected in step (f) is indicative that the test compound is an antagonist of the known effect.
In one embodiment of the method, if step (f) indicates that the compound is an agonist of the known effect, then the method includes automatically determining the concentration dependence of agonist activity of the test compound by repeating steps (a), (b), and (c), and (d), (e), and (f) while varying the concentration of the test compound and the standard compound and recording resultant changes in the cellular response; and if step (f) indicates that the compound is an antagonist, then the method includes automatically determining the concentration dependence of inhibition of the cellular response in the presence of the agonistic standard compound by repeating steps (d), (e), and (f) while varying the concentration of the test compound and the standard compound and recording resultant changes in the cellular response. Optionally, the method also comprises the step, when step (f) indicates that the compound is an antagonist, of: (g) automatically determining the concentration dependence of cell response activation by repeating steps (d), (e), and (f) for a zero concentration of the test compound while varying the concentration of the standard compound and recording resultant changes in cellular response, and then repeating this step (g) for different concentrations of the test compound. The method may further include the step when step (f) indicates that the compound is an agonist, of: (h) automatically determining the concentration dependence of cell response activation by repeating steps (d), (e), and (f) for a zero concentration of the standard compound while varying the concentration of the test compound and recording resultant changes in cellular response, and then repeating this step (h) for different concentrations of the compound. Variation of the concentration of the test compound and/or the standard compound can be done continuously or in a stepwise manner. One preferred step includes graphically displaying the recorded changes in the cellular response.
The cellular response can be any desired cellular response susceptible of being measured or detected as the cells flow past a detector in suspension. It can be evidenced by analyzing the cells themselves or the medium in which the cells are suspended. Cellular responses can be measured, for example, from a change in intracellular ion concentration, such as calcium, magnesium, proton, sodium, or potassium. In one embodiment, the ion is detected using an intracellular dye such as a visible and/or fluorescent dye.
In another embodiment, a method comprises the steps of: (a) combining a homogeneous suspension of living cells with a test compound having an unknown cellular effect to form a test mixture, (b) directing the test mixture through a detection zone; (c) measuring a cellular response of the suspended cells to the test compound as the test mixture is flowing through the detection zone; and repeating steps (a)-(c) on each cell type of a series of cell types to be tested so as to measure the effect of said test compound on each cell type of said series of cell types. In this embodiment, the method may further comprise the steps of: combining a homogenous suspension of each of said cell types in said series of cell types with a standard compound having a known effect on said cellular response of said cells to form a series of standard mixtures; directing said series of standard mixtures through the detection zone; and measuring the cellular response of each of said cell types to said standard compound. The standard compound may be an agonist or antagonist of said cellular response.
The invention also includes an apparatus for automatically measuring the effect of a plurality of test compounds on living cells, comprising: a test compound sampler for sequentially providing samples of multiple test compounds, a cell suspension input for providing a homogeneous suspension of living cells, a mixing zone, coupled to the test compound sampler, for receiving the samples of the test compounds from the test compound sampler, and receiving the suspension of living cells from the cell suspension input and mixing each test compound with the suspension of living cells; and a detector, coupled to the mixing zone, for measuring a cellular response of the suspended cells to each test compound. The apparatus may additionally include a standard compound sampler, coupled to the mixing zone, for providing a sample of a standard compound having a known effect on the cellular response of the suspended cells, wherein the mixing zone receives the sample of the standard compound from the standard compound sampler and mixes the standard compound with the suspended cells and the detector measures the cellular response of the suspended cells to the standard compound. In one embodiment, the mixing zone simultaneously mixes the test compound and the standard compound with the suspended cells and the detector detects the known effect or an alteration of the known effect. The apparatus may also include a first gradient device, coupled to the test compound sampler, for automatically adjusting the concentration level of the test compound transferred to the mixing zone from the test compound sampler; and a second gradient device, coupled to the standard compound sampler, for automatically adjusting the concentration level of the standard compound transferred to the mixing zone from the standard compound sampler. The apparatus may further include a switching valve, coupled to the first and second gradient devices at an input of the switching valve and coupled to the mixing zone at an output of the switching valve, for selectively switching the flow of a concentration of the test compound or a concentration of the standard compound or both to the mixing zone where the test compound and/or the standard compound is then mixed with the suspension of cells. In addition, the apparatus may include a calibration unit, coupled to the switching valve, wherein the switching valve also selectively switches the flow of a calibration solution provided by the calibration unit into the mixing zone where the calibration solution is mixed with the suspension of cells.
The reaction time of the cells with the various test and standard compounds may be controlled through use of various lengths of reaction developing lines coupled to the output of the mixing zone, for receiving a mixture of the cell suspension mixed with either the test compound, the standard compound or the calibration solution, and providing a flow path for the mixture such that there is adequate time for the suspension cells to react with the test compound, the standard compound or the calibration solution, wherein the reaction developing lines is further coupled to the input of the detector which receives the mixture from the reaction developing lines. In one preferred embodiment, the detector detects changes in intracellular ion concentration. Preferred ions are described above.
The apparatus may additionally include a controller, coupled to the first and second gradient devices, the test compound sampler, the standard compound sampler and the switching valve, for controlling their operation; and a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to the detector in order to send and receive cell response measurement signals to and from the detector. In order to automate the apparatus further, the test compound sampler can be an automated robotic sampler capable of selecting a specified test compound from a library of test compounds. A controller can be coupled to the test compound sampler, for controlling the operation of the test compound sampler, using the computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, thereby controlling the selection and retrieval of test compounds by the test compound sampler from the test compound library.
The apparatus can direct flow through the various elements under either positive or negative pressure. Thus, one embodiment includes a gradient pump having an input and an output, coupled to the test compound sampler, for adjusting the concentration level of the test compound transferred to the mixing zone from the test compound sampler, wherein the test compound sampler comprises a first intake nozzle for receiving the specified test compound, a second intake nozzle for receiving a buffer solution; and wherein the gradient pump is coupled to the first and second intake nozzles and receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound. This embodiment may also include a standard compound sampler for providing a sample of a standard compound to the mixing zone. The standard compound sampler is preferably an automated robotic sampler capable of selecting a specified standard compound from a library of standard compounds. The apparatus may also include a second gradient pump having an input and an output, coupled to the standard compound sampler, for adjusting the concentration level of the standard compound provided to the mixing zone from the standard compound sampler, wherein the standard compound sampler comprises a third intake nozzle for receiving the specified standard compound, a fourth intake nozzle for receiving a buffer solution; and wherein the second gradient pump is coupled to the third and fourth intake nozzles and receives specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound. The apparatus may further comprise a second mixing zone coupled to the outputs of the first and second gradient pumps, for receiving and mixing the specified concentrations of the specified test compound and the specified standard compound, such that the output of the second mixing zone is provided to the first mixing zone. Another element that can be included is a calibration unit for providing a calibration solution; and a switching valve, having a first input coupled to the second mixing zone, a second input coupled to the calibration unit, and an output coupled to the first mixing zone, for switching between the flow of either a compound mixture from the second mixing zone or the calibration solution from the calibration unit and then providing the flow to the first mixing zone where it may be mixed with the cell suspension. Preferably, the calibration unit comprises a calibration maximum solution which provides for maximal cell response when mixed with the cell suspension, a calibration minimum solution which provides for minimal cell response when mixed with the cell suspension, a diverting valve having a first input coupled to the calibration maximum solution and a second input coupled to the calibration minimum solution, for switching between the flow of either the calibration maximum solution or calibration minimum solution; and a pump, coupled to the output of the diverting valve and an input of the switching valve, for pumping either the calibration maximum or calibration minimum solution from the diverting valve into the switching valve. The positive pressure version of the apparatus can also include a second pump, coupled to an input of the first mixing zone, for pumping the suspension of cells from the cell suspension input into the first mixing zone. Reaction developing lines, having an input coupled to an output of the first mixing zone and an output coupled to an input of the detector, for providing a flow path and a reaction time delay for a mixture received from the first mixing zone and for providing the mixture to the detector, can also be included.
In a preferred embodiment, as above, the apparatus can include a controller, coupled to the first and second gradient pumps, the test compound sampler, the standard compound sampler and the switching valve, the first and second mixing zones, the first and second pumps and the diverting valve for controlling their operation; and a computer, coupled to the controller, for sending command signals to the controller in accordance with a software program implemented by the computer, wherein the computer is also coupled to the detector in order to send and receive cell response measurement signals to and from the detector.
The apparatus of the present invention can also be run under negative pressure by utilizing a pump, coupled to the output of the detector, for providing negative pressure to the apparatus, a proportionating valve, coupled to the test compound sampler, for adjusting the concentration level of the test compound transferred to the mixing zone from the test compound sampler, wherein the test compound sampler further comprises a first intake nozzle for receiving the specified test compound, a second intake nozzle for receiving a buffer solution; and the proportionating valve receives specified concentrations of the test compound by adjusting the amount of test compound and buffer solution received by the first and second intake nozzles, respectively, wherein the buffer solution is a diluting agent of the test compound. The negative pressure apparatus can also include an automated standard compound sampler capable of selecting a specified standard compound from a library of standard compounds, the standard compound sampler including a third intake nozzle for receiving the specified standard compound and a fourth intake nozzle for receiving a buffer solution; and a second proportionating valve, coupled to the third and fourth intake nozzles, for receiving specified concentrations of the standard compound by adjusting the amount of standard compound and buffer solution received by the third and fourth intake nozzles, respectively, wherein the buffer solution is a diluting agent of the standard compound. In one version, the apparatus includes a first priming valve, coupled to the output of the first proportionating valve, for receiving the specified concentration of the test compound and providing the test compound to the mixing zone, and a second priming valve, coupled to the output of the second proportionating valve, for receiving the specified concentration of the standard compound and providing the standard compound to the mixing zone. A calibration unit can be included as described previously, as can reaction developing lines, robotic input, and computer control. The cell suspension input may comprise a cell suspension reservoir, a buffer reservoir, a third diverting valve, having a first input coupled to the cell suspension reservoir and a second input coupled to the buffer reservoir, for adjusting the concentration of the cell suspension, wherein the buffer is a diluting agent of the cell suspension, and a fourth priming valve, coupled to the output of the third diverting valve, for receiving the cell suspension mixture from the third diverting valve and providing this mixture to the second mixing zone. In one embodiment, the apparatus further comprises a plurality of cell suspension reservoirs.
Another aspect of the present invention is a method of characterizing the receptors present in a cell comprising the steps of (a) combining a suspension of cells with a test agent known to influence the activity of a particular receptor to form a test mixture; (b) directing said test mixture through a detection zone; (c) measuring a cellular response of said suspension of living cells to said test agent as said test mixture is flowing through said detection zone wherein a response to a test agent indicates that said cell expresses a receptor known to respond to said test agent; and (d) repeating steps (a)-(c) with a series of test agents until the effects of each test agent has been measured. The test agent may comprise an agonist, an antagonist, or a mixture of an antagonist and an agonist. In one embodiment, this method further comprises repeating steps (a)-(d) on a series of different cell types to determine the receptors expressed by each cell type.
Another aspect of the invention is a method of confirming that a test compound has an effect on the activity of a receptor comprising the steps of contacting a negative control cell type which lacks said receptor with said test agent to form a negative control mixture; directing said negative control mixture through a detection zone; measuring the cellular response of said suspension of living cells to said test compound as said negative control mixture is flowing through said detection zone; contacting cells of the same cell type as the negative control which have been engineered or induced to express said receptor with said test agent to form a test mixture; directing said test mixture through a detection zone; measuring the cellular response of the cells in the test mixture to said test agent, whereby a difference in the measured response of said cells in said test mixture relative to the measured response of said negative control cells indicates that said test agent has an effect on the activity of said receptor. The test agent may comprise an agonist, an antagonist, or a mixture of an agonist and an antagonist.
Another aspect of the present invention is a method of determining the activity of one or more receptors in a series of cell types comprising (a) combining a suspension of living cells comprising a member of said series of cell types with an agent known to influence the activity of a particular receptor to form a test mixture; (b) directing the test mixture through a detection zone; (c) measuring the cellular response of said suspension of living cells to said test agent; (d) repeating steps (a)-(c) on each member of the series of cell types until the effect of said test agent has been measured in each cell type of said series. The test agent may comprise a known receptor agonist, a known receptor antagonist a mixture of a known agonist and a known antagonist, a compound whose activity is unknown, or a mixture of a compound whose activity is unknown and a compound which is a known agonist or a known antagonist.
Certain preferred embodiments of the present invention are discussed below in more detail in connection with the drawings and the detailed description of the preferred embodiments. These preferred embodiments do not limit the scope or nature of the present invention.